Lipids when dispersed in aqueous media readily form liposomes, such as unilamellar vesicles and multilamellar vesicles. Liposomes have been used successfully to encapsulate and deliver a wide range of chemicals including nucleic acids, proteins and small molecule drugs, to cells.
Cationic liposomes prepared from a composition of cationic lipids and phospholipids, readily form aggregates with anionic macromolecules such as DNA and RNA. These cationic liposome—nucleic acid aggregates are often engineered such that the net charge of the complex is positive, which is believed to facilitate interaction with the anionic cell surface thereby enhancing uptake of the encapsulated cargo and subsequent cell transfection. An example of a cationic lipid composition that is commonly used for the transfection of cells in vitro is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) combined with dioleoylphosphatidylethanolamine (DOPE) at a molar ratio of 1:1.
The size and structure of cationic liposome—nucleic acid aggregate is dependent on the lipid composition and the method of manufacture. These structures can range in size from several hundred nanometers to micrometers and often have heterogeneous morphologies when visualized by electron microscopy, including the classic “spaghetti and meatballs” conformation.
Cationic liposome—nucleic acid aggregates have limited effectiveness in primary cells, i.e., cells harvested from a living organism. This is believed to be the result of toxicity due to excessive cationic charge. Toxicity plus large particle size also limits use of cationic liposome—nucleic acid aggregates for transfection in vivo.
Lipid nanoparticles (LNP) are the most clinically advanced drug delivery systems, with seven LNP-based drugs having received regulatory approval. These approved drugs contain small molecules such as anticancer drugs and exhibit improved efficacy and/or reduced toxicity compared to the “free” drug. LNP carrier technology has also been applied to delivery of “genetic” anionic macromolecules such as plasmids for protein expression or small interfering RNA (siRNA) oligonucleotides (OGN) for gene silencing.
Recent advances in LNP technology and the cationic lipids used to encapsulate and deliver of genetic drugs, have enabled siRNA-LNP that have been shown to overcome the inherent liabilities of cationic liposome—nucleic acid aggregates and mediate silencing of therapeutically relevant target genes in difficult-to-transfect primary cells and animal models, including non-human primates following intravenous (i.v.) injection. These siRNA-LNP are currently under evaluation in several clinical trials.
A variety of methods have been developed to formulate LNP systems containing genetic drugs. These methods include mixing preformed LNP with OGN in the presence of ethanol, or mixing lipid dissolved in ethanol with an aqueous media containing OGN, and result in LNP with diameters of 100 nm or less and OGN encapsulation efficiencies of 65-95%. Both of these methods rely on the presence of cationic lipid to achieve encapsulation of OGN and poly(ethylene glycol) (PEG) lipids to inhibit aggregation and the formation of large structures. The properties of the LNP systems produced, including size and OGN encapsulation efficiency, are sensitive to a variety of formulation parameters such as ionic strength, lipid and ethanol concentration, pH, OGN concentration and mixing rates. In general, parameters such as the relative lipid and OGN concentrations at the time of mixing, as well as the mixing rates are difficult to control using current formulation procedures, resulting in variability in the characteristics of LNP produced, both within and between preparations.
Microfluidic devices rapidly mix fluids at the nanoliter scale with precise control over temperature, residence times, and solute concentrations. Controlled and rapid microfluidic mixing has been previously applied in the synthesis of inorganic nanoparticles and microparticles, and can outperform macroscale systems in large-scale production of nanoparticles. Microfluidic two-phase droplet techniques have been applied to produce monodisperse polymeric microparticles for drug delivery or to produce large vesicles for the encapsulation of cells, proteins, or other biomolecules. The use of hydrodynamic flow focusing, a common microfluidic technique to provide rapid mixing of reagents, to create monodisperse liposomes of controlled size has been demonstrated. This technique has also proven useful in the production of polymeric nanoparticles where smaller, more monodisperse particles were obtained, with higher encapsulation of small molecules as compared to bulk production methods.
Despite the numerous products available for cell transfection, a need exists for devices and methods for the efficient delivery of siRNA OGN and other anionic macromolecules to difficult-to-transfect primary cells in vitro and to target cells in vivo. The present invention seeks to fulfill this need and provides further related advantages to address a major problem impeding the validation of aberrant genes, identified through genome sequencing of disease cells, as potential drug or biomarker targets.